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Vol. 63, Issue 1, 183-191, January 2003
Laboratory of Molecular Physiology (P.J.K., S.R.I.) and Laboratory of Integrative Neuroscience (M.I.D.), National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Metabotropic glutamate receptor 2 (mGluR2) is a class 3 G
protein-coupled receptor and an important mediator of synaptic activity in the central nervous system. Previous work demonstrated that mGluR2
couples to pertussis toxin (PTX)-sensitive G proteins. However, the
specificity of mGluR2 coupling to individual members of the
Gi/o family is not known. Using heterologously expressed mGluR2 in rat sympathetic neurons from the superior cervical ganglion (SCG), the mGluR2/G protein coupling profile was characterized by
reconstituting coupling in PTX-treated cells expressing PTX-insensitive mutant G
proteins and G
. By employing this method, it was
demonstrated that mGluR2 coupled strongly with Gob,
Gi1, Gi2, and Gi3, although coupling to Goa was less efficient. In
addition, mGluR2 did not seem to couple to the most divergent member of
the Gi/o family, Gz, although
Gz coupled strongly to the endogenous 2 adrenergic
receptor. To determine which G proteins may be natively expressed in
SCG neurons, the presence of mRNA for various G proteins was tested
using reverse transcription-polymerase chain reaction. Strong bands
were detected for all members of the Gi/o family
(Go, Gi1, Gi2,
Gi3, Gz) as well as for
G11 and Gs. A weak signal was detected
for Gq and no G15 mRNA was detected.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Metabotropic
glutamate receptors (mGluRs) are members of the class 3 G
protein-coupled receptor family, which includes the calcium sensing
receptor and the GABAB receptor, among others (Conn and Pin, 1997
). There are eight known mammalian mGluR genes (mGluR1-8), which play diverse roles in the nervous system, including the modulation of synaptic transmission from both pre- and postsynaptic locations and regulation of synaptic plasticity. In addition, mGluRs
also play a role in mediating sensory transduction (Bortolotto et al.,
1994; Conn and Pin, 1997; Wilsch et al., 1998). mGluRs have been
divided into three groups based on sequence homology, sensitivity to
pharmacological agents, and G protein-coupling specificity (De Blasi et
al., 2001). Group II (mGluRs 2 and 3) and group III mGluRs (mGluRs 4, 6-8) are known to couple exclusively to the pertussis toxin
(PTX)-sensitive Gi/o family of G proteins (Tanabe
et al., 1992, 1993; Saugstad et al., 1994), whereas group I mGluRs
couple to multiple classes of G proteins (Abe et al., 1992; Aramori and
Nakanishi, 1992; Pin et al., 1992; Joly et al., 1995).
The mechanism of mGluR/G protein coupling has been examined in several
studies (Pin et al., 1995; Gomeza et al., 1996; Blahos et al., 1998;
Mary et al., 1998). Clearly, activation of G proteins by mGluRs in
response to agonist binding involves regions of the receptor that are
distinct from those of the class 1 G protein-coupled receptors.
Coupling of mGluRs to G proteins seems to involve the proximal end of
the intracellular C-terminal tail (Mary et al., 1998) and part of the
second intracellular loop (Pin et al., 1995; Gomeza et al., 1996).
Chimeric group II/group I mGluRs in which these regions from a group I
mGluR were inserted into mGluR3 were able to couple to phospholipase C
(similar to wild-type group I mGluRs; Gomeza et al., 1996). In
addition, residues on the C-terminal tail of group I mGluRs seem to be
involved in coupling to Gq/11, because this
region has been shown to participate in phospholipase C activation
(Mary et al., 1998). Thus, although many studies have examined the
molecular basis of mGluR coupling to distinct G protein families
(Gomeza et al., 1996; Blahos et al., 1998), detailed studies of the G
protein coupling specificity of an mGluR within a single G
protein family have not been performed. Such studies may begin to shed
light on the molecular basis for specificity in systems such as
synaptic terminals in the central nervous system, where several types
of G protein-coupled receptor are present.
PTX is a valuable tool for the study of heterotrimeric G proteins. By
ADP-ribosylating the last cysteine residue in the extreme C terminus of
Gi/o proteins (present only on G proteins
in this family), PTX treatment selectively uncouples these G proteins (Milligan, 1988). Consequently, mutation of this cysteine to another residue renders the resulting G PTX-insensitive. Therefore, after treating cells expressing a given Gi/o-coupled
receptor with PTX to inactivate endogenous
Gi/o proteins, the G protein-coupling specificity of the receptor to G proteins within this family can be
examined by heterologously expressing individual G CG or CI mutants
and examining coupling. This method has been used to examine the
coupling of other G protein-coupled receptors (Taussig et al., 1992;
Senogles, 1994; Wise et al., 1997; Jeong and Ikeda, 2000).
In this study, the G protein specificity of mGluR2 for G proteins in
the Gi/o family was examined by reconstituting
mGluR coupling in PTX-treated cells through expression of
PTX-insensitive G (C-terminal Cys to Gly) mutants in sympathetic
neurons from the rat superior cervical ganglion (SCG). In addition, to
determine which subtypes of G proteins may be endogenously expressed
in SCG neurons, the presence of mRNA for nine different G subunits was determined using RT-PCR.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Isolation, DNA Injection, and Plasmids.
A detailed
description of the cell isolation and cDNA injection protocol is
published elsewhere (Ikeda, 1997). The animal protocols used were
approved by the Institutional Animal Care and Use Committee. Briefly,
both SCGs were removed from adult Wistar rats (175-225 g) after
decapitation, and incubated in Earle's balanced salt solution
(Invitrogen, Carlsbad, CA) containing 0.45 mg/ml trypsin
(Worthington Biochemicals, Freehold, NJ), 0.6 mg/ml collagenase D
(Roche Applied Science, Indianapolis, IN), and 0.05 mg/ml DNase
I (Sigma Chemical, St. Louis, MO) for 1 h at 35°C. Cells were
then centrifuged (50 g), transferred to minimum essential medium
(Fisher Scientific, Pittsburgh, PA), plated on
poly(L-lysine)-coated 35-mm polystyrene tissue culture
dishes and incubated (95% air/5% CO2; 100%
humidity) at 37°C before DNA injection. After injection, cells were
incubated overnight at 37°C and patch-clamp experiments were
performed the following day. Where indicated, neurons were incubated
overnight with PTX (0.5 µg/ml; List Biological, Campbell, CA) in the
culture media.
20°C as a 1 µg/µl stock solution in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8). Rat mGluR2
was injected at 50 ng/µl (pCI; Promega, Madison, WI). Construction of
the PTX-insensitive mutants of Gi1-3 and
Goa,b has been described previously (Jeong and
Ikeda, 2000). For reconstitution experiments, all G cDNAs (pCI;
Promega) were injected at 5 to 6 ng/µl with bovine G1 and G2
(from M. I. Simon, Howard Hughes Medical Institute, California
Institute of Technology, Pasadena, CA) injected at 10 ng/µl
each (pCI; Promega). Neurons were coinjected with "enhanced" green
fluorescent protein cDNA (0.005 µg/µl; pEGFP-N1; BD Clontech
Laboratories) to facilitate later identification of successfully
injected cells.
All inserts were sequenced using an automated DNA sequencer (ABI 310;
Applied Biosystems, Foster City, CA). PCR products were purified with
QIAGEN (Valencia, CA) silica membrane spin columns before restriction
digestion and ligation. Plasmids were propagated in XL1-blue bacteria
(Stratagene, La Jolla, CA) and midipreps prepared using QIAGEN anion
exchange columns.
Electrophysiology and Data Analysis.
Patch pipettes were
made from 7052 glass (Garner Glass, Claremont, CA) and had resistances
of 1 to 4 M
. Series resistances were 2 to 6 M before electronic
compensation, which was typically 
80%. Ruptured patch whole-cell
recordings were made with an Axopatch 200A patch-clamp amplifier (Axon
Instruments, Union City, CA). Voltage protocol generation and data
acquisition were performed using custom software on a Macintosh Quadra
series computer (Apple Computer, Cupertino, CA) with a MacADIOS II data
acquisition board (G.W. Instruments, Somerville, MA). Currents were
low-pass-filtered at 5 kHz using the four-pole Bessel filter in the
patch-clamp amplifier, digitized at 2 to 5 kHz and stored on the
computer for later analysis. Experiments were performed at 21 to 24°C
(room temperature). Data analysis was performed using Igor software (Wavemetrics, Lake Oswego, OR).
250 ms). The degree
of mGluR-mediated calcium current inhibition (and
norepinephrine-mediated inhibition, where indicated) was calculated as
the maximal inhibition of the current in the presence of drug compared
with the last current measurement before application of the drug.
RT-PCR.
To test for the presence of mRNA coding for each of
the nine G subunits (i1-3, o, z, q, 11, s, and 15), unique 18- to
25-base primer pairs from coding or 3' noncoding sequences were
identified using MacVector software (Accelrys, Inc., Princeton, NJ).
Potential primers were constrained by length, GC content, melting
temperature, and product size (see Table
1 for primer list and expected product sizes). Where possible, longer PCR products that were more likely to
span introns were selected to reduce the contribution of genomic DNA.
In addition, samples were treated with DNase as part of the RNA
isolation procedure. Each potential primer was then BLAST-searched to
rule out the presence of homologous or identical sequences present in
other known rat mRNAs. The primer sets for each G, as well as the
size (number of bases) of the expected product are shown in Table 1.
RT-PCR was performed using the QIAGEN One-Step RT-PCR kit on 40 ng of
isolated total RNA (annealing temperature of 60-63°C, for 30-35
cycles) from dissociated rat SCG cultures using the Qiagen RNeasy total
RNA isolation kit. As a positive control, primers were constructed to
detect the ubiquitously expressed glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) message (5'- CCAAAAGGGTCATCATCTCCG-3', and 5'-
AGACAACCTGGTCCTCAGTGTAGC-3', producing an expected product of 501 bases). Negative controls were performed with the GAPDH primers in the
absence of RNA. Primers were obtained from Operon Technologies
(Alameda, CA). RT-PCR products were run on 3% agarose precast gels
from Bio-Rad (Hercules, CA).
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Western Blotting.
Homogenates (10%, w/v) were made from
combined SCGs dissected from an adult rat, and protein concentrations
were determined using the bicinchoninic acid assay (Pierce
Biotechnology, Rockford, IL). Polyacrylamide gels (10%) were used for
protein fractionation and parallel gels were stained with Coomassie
blue to verify loading of proteins, separation, and sample integrity.
Proteins were then transferred to polyvinyl difluoride membranes for
immunodetection. The membranes were blocked for 1 h with 5% powdered
milk in 25 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, then probed
with anti-G antibodies at dilutions in the same medium. Antibodies used were anti-Gz [1:200, 4°C overnight;
Santa Cruz Biotechnology (Santa Cruz, CA) and Calbiochem (San Diego,
CA)] and anti-Gs (1:5,000, 1 h, 22°C;
PerkinElmer Life Sciences, Boston, MA),
anti-Gq/11 (1:10,000, 1 h, room
temperature; Calbiochem), polyclonal anti-Gi3 (Calbiochem) and monoclonal anti-Gi2 (both at
1:1000, 4°C overnight; LabVision, Fremont, CA). Horseradish
peroxidase-conjugated donkey anti-rabbit (Cell Signaling Technology,
Beverly, MA) was used (1:1000) for detection. Peroxidase activity was
detected with SuperSignal West Pico (Pierce Biotechnology) and
visualized using a Kodak Image Station 1000 or with X-ray film (XAR-2;
Eastman Kodak, Rochester, NY).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Calcium Current Inhibition by Heterologously-Expressed mGluR2.
Neurons isolated from the rat SCG do not functionally express mGluRs
(Ikeda et al., 1995). Heterologous expression of distinct subtypes of
mGluRs in SCG neurons is therefore a useful model system for studying
the properties of individual mGluR subtypes. Cells expressing mGluR2
after intranuclear cDNA injection respond to application of 100 µM
L-glutamate with a fast and potent inhibition of the
predominantly N-type (Zhu and Ikeda, 1994) whole-cell calcium current
(Ikeda et al., 1995; Kammermeier et al., 2000). The time course of this
inhibition is illustrated in Fig. 1A (see
inset for samples of control and Glu-inhibited current traces). The `triple-pulse' voltage protocol (Elmslie et al., 1990) was used to
illustrate the voltage-dependent nature of the modulation and to
measure basal facilitation as an indicator of free G levels (see
below). The average magnitude of mGluR2-mediated calcium current
inhibition was 59 ± 2% (n = 31; Fig. 1C). Cells
expressing mGluR2 and treated overnight with 500 ng/ml PTX did not
exhibit any detectable calcium current inhibition in response to Glu
application (Fig. 1B). Calcium current inhibition in PTX-treated cells
was 2 ± 0.3% (n = 24; Fig. 1C). This result
confirms previous observations that mGluR2 couples to PTX-sensitive,
Gi/o G proteins (Chavis et al., 1994; Ikeda et
al., 1995).
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Reconstitution of G Protein Coupling after PTX Treatment.
The
strategy for examining the specificity of mGluR2 coupling to
Gi/o proteins is illustrated in Fig.
2A. First, cells were intranuclearly
injected with cDNA for mGluR2 plus G1,
G2 (this G combination was chosen for
its ability to robustly modulate N-type calcium currents when expressed
in SCG neurons), and a PTX-insensitive mutant (or naturally
PTX-insensitive wild-type) G. Next, cells were treated overnight
with PTX to inactivate endogenous Gi/o
proteins. Finally, calcium current facilitation was examined to
determine the G/G stoichiometry.
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-mediated and voltage-dependent (Herlitze et al., 1996; Ikeda, 1996). This is evident from the slowed activation kinetics of the
inhibited currents and from the `facilitation' observed after a
strong depolarizing prepulse (Bean, 1989; Elmslie et al., 1990) (Fig.
1A, inset). These features are hallmarks of the G-mediated calcium current inhibitory pathway. Commonly, facilitation is defined
as the current in the postpulse divided by the current at the same time
in the prepulse (the first test pulse to +10 mV). Thus, facilitation
can be used as a quantitative measure of relative free G levels
in the cell. Overexpression of G alone mimics this modulation and
produces basal currents with slow activation and strong basal
facilitation (
1) (Ikeda, 1996; Garcia et al., 1998; Ruiz-Velasco
and Ikeda, 2000). Overexpression of G subunits alone produces basal
currents with facilitation <1, because of strong buffering of
endogenously expressed G. Under these conditions, agonist-induced
G-mediated calcium current modulation is occluded.
As illustrated in Fig. 2B, heterologous expression of G and G
resulted in cells with currents that were placed into three functional
categories. In the first category were placed all cells that exhibited
strong basal facilitation (> 1.3, chosen arbitrarily because basal
facilitation this high was rare in control cells in this study: 1 of 55 cells). This level of facilitation was an indication of excess free
G. The second category included those cells with basal
facilitation < 1, indicating excess G, resulting from G
buffering by expressed G. The third category included cells with
basal facilitation between 1 and 1.3, indicating a good functional
stoichiometric balance of G and G. Therefore, G/G-expressing cells with basal facilitation in this range were chosen for analysis (Jeong and Ikeda, 2000). In addition to
determining stoichiometric balance, these criteria provide a control
for expression levels of different G subunits, assuming that levels
of G12 remain
relatively constant.
mGluR2 Coupling to Gi/o G Proteins.
After
treatment with PTX, cells expressing mGluR2 exhibited no detectable
calcium current inhibition in response to 100 µM L-glutamate (Glu; as described in Fig. 1B). Over this
background, PTX-insensitive Gi/o proteins [with
a Cys-to-Gly mutation in the extreme C terminus, denoted GC351G (or
GC352G)] were expressed with G to reconstitute coupling and
examine the specificity of mGluR2/G protein interactions. Calcium
current inhibition in PTX-treated cells expressing
GobC351G (and
G12) was strong (Fig. 3A, a), indicating that GluR2
couples efficiently to Gob. The magnitude of
calcium current inhibition in these cells was indistinguishable from
paired control cells (recorded the same days; Fig. 3B, 
). Calcium
currents in PTX-treated cells reconstituted with
GobC351G were inhibited 66 ± 5%
(n = 5) upon Glu application. Calcium currents in
paired control cells (PTX-untreated) were inhibited 68 ± 4%
(n = 6), whereas currents in paired PTX-treated control
cells were inhibited 0.5 ± 0.4% (n = 6).
Dose-response curves for calcium current inhibition in control and
GobC351G-reconstituted cells are illustrated
in Fig. 3A, b. EC50 values for the two groups, determined by fitting to a single-site binding isotherm equation (see
Fig. 3 legend), were comparable. Control cells had an
EC50 of 1.3 µM, compared with 3.8 µM for
GobC351G-reconstituted cells. These data
provide evidence that the CG mutation in the C terminus of the G
subunit did not detectably alter the receptor-G protein interaction.
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oaC351G was less
efficient, exhibiting calcium current inhibition of only 27 ± 10% (n = 7) compared with 54 ± 5%
(n = 6) in control cells from the same experimental days (Fig. 3B). PTX-treated control cells from the same preparations were inhibited 2 ± 1% (n = 6). These data
indicate that mGluR2 couples more efficiently to
GobC351G than to
GoaC351G, a surprising result because the
extreme C terminus, a region demonstrated to be critical in
receptor/G interaction (Hamm et al., 1988; Conklin et al., 1993), is
nearly identical in these two splice variants. Poor expression of the
GoaC351G construct cannot sufficiently explain
these results because G expression levels were balanced with G
expression. Finally, mGluR2 seemed to be unable to couple to
Gz. Calcium current inhibition in PTX-treated
cells reconstituted with Gz (a naturally
PTX-insensitive member of the Gi/o family) was
virtually undetectable at only 3 ± 2% (n = 6),
compared with 61 ± 4% (n = 3) in PTX-untreated
cells, and 1 ± 0.6% (n = 3) in PTX-treated
control cells (Fig. 3B).
As negative controls, similar reconstitution experiments were performed
using wild-type Gq or wild-type
Gob (Fig. 4). As expected, no detectable calcium current inhibition was evident in
PTX-treated cells expressing either Gq, which
is not coupled to mGluR2, or Gob, the
PTX-sensitive wild-type G. Stoichiometrically balanced PTX-treated
cells coexpressing mGluR2,
G12, and
Gq were not inhibited by Glu (0 ± 0.3%,
n = 5), compared with inhibitions of 61 ± 4%
(n = 3) in PTX-untreated paired control cells and
1 ± 0.6% (n = 3) in PTX-treated paired control
cells. Similarly treated cells coexpressing mGluR2,
G12, and wild-type
Gob were inhibited 1 ± 2%
(n = 2) by Glu, compared with 47 ± 12%
(n = 3) and 3 ± 0.2% (n = 3) in
PTX-untreated and -treated control cells, respectively.
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z was
coexpressed with G12
to reconstitute coupling to the natively expressed
2 adrenergic receptor in PTX-treated cells.
Coupling of the 2 adrenergic receptor to
Gz in SCG neurons has been demonstrated
previously (Jeong and Ikeda, 1998). In PTX-treated cells coexpressing
G and Gz (in functional stoichiometric
balance), 10 µM norepinephrine (NE) inhibited calcium currents
69 ± 3% (n = 3). Paired PTX-untreated and
-treated cells were inhibited 74 ± 1% (n = 3)
and 13 ± 5% (n = 3), respectively. These data
demonstrate that Gz is expressed and is
capable of coupling a G protein-coupled receptor to calcium channels in
SCG neurons.
Finally, the remaining members of the Gi/o family
were examined. Figure 5A, inset,
illustrates the time course and sample currents from a PTX-treated,
mGluR2-expressing cell reconstituted with G and
Gi2C352G. Glu-mediated calcium current
inhibition in this cell was potent, indicating that mGluR2 couples
efficiently to Gi2 in this system. On average,
PTX-treated cells whose mGluR2 coupling was reconstituted with
Gi2C352G were inhibited 48 ± 10%
(n = 8) by Glu, compared with 53 ± 6%
(n = 5) and 2 ± 1% (n = 3) in
paired PTX-untreated and -treated control cells, respectively (Fig.
5B). In addition, mGluR2 seemed to be similarly capable of coupling to
Gi1 and Gi3 (Fig.
5B). Reconstitution using the CG mutants of these G proteins also
exhibited efficient coupling to calcium currents. PTX-treated cells
coexpressing mGluR2, Gi1C352G, and
G12 were inhibited
57 ± 9% (n = 5) by Glu. Inhibition in PTX-untreated, paired control cells was 68 ± 2%
(n = 4) and in PTX-treated, paired control cells
inhibition was 2 ± 2% (n = 3). PTX-treated cells
coexpressing mGluR2, Gi3C352G, and
G12 were inhibited
68 ± 1% (n = 4) by Glu. Inhibition in
PTX-untreated, paired control cells was 51 ± 7%
(n = 4), and in PTX-treated, paired control cells,
inhibition was 3 ± 1% (n = 3).
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Endogenous Expression of G Proteins in SCG Neurons.
To
determine which G proteins may be expressed natively in SCG neurons,
and to shed some light on possible receptor/G protein interactions of
natively-expressed receptor/G protein pairs (or in the case of mGluR2,
heterologously expressed receptor/native G protein pairs), RT-PCR was
used to detect mRNA for several G proteins. Unique primer sets were
designed for several G proteins, including o, i1-3, z, q, 11, s,
and 15. In addition, primers for the housekeeping gene GAPDH were used
as a positive control. Table 1 lists the primer sequences and predicted
product size for each G primer set. Figure
6A shows the results of RT-PCR reactions targeting each of the Gi/o G proteins. In each
case, a clear band at the predicted product size was detected. In
addition, the GAPDH positive and negative (no RNA) controls are shown.
These data suggest that SCG neurons may potentially express each member of the Gi/o G protein family.
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proteins, the
presence of message for several other G proteins was tested using
RT-PCR. As Fig. 6B indicates, bands were detected at the predicted
product sizes for Gq,
G11, and Gs. However,
the Gq band seemed much weaker than that of
the other G proteins. This suggests that the
Gq mRNA is unstable or perhaps present at
lower levels than the other G proteins tested, but poor
hybridization by the selected primers is the more likely cause of the
weak signal. Therefore, one can only infer that
Gq message is present in rat SCGs. In
addition, previous studies have confirmed the presence of
Gq in SCG neurons, as well as
Go and G11 (Haley et
al., 1998). Finally, G15 message was
undetectable in RNA from SCG neurons. This result was expected because
G15 expression is confined to hematopoietic
cells (Wilkie et al., 1991).
Western blotting was used to confirm the results of RT-PCR experiments
where specific antibodies were available, as judged by detection of
recombinant proteins (Fig. 6C). Of the antibodies tested, only three
(anti- Gi2,
anti-Gq/11, and
anti-Gs) seemed specific as judged by
detection of recombinant proteins (see Fig. 6C). Others, namely
Gi1, Gi3, and
Go, detected protein from SCG near the
appropriate molecular weight, but also detected at least one
inappropriate recombinant control (data not shown), so the presence of
specific proteins could not be determined with confidence. The positive
result for Gz RNA was surprising. Previously, Gz protein has been shown to be present in
brain and few other tissues at low levels (Fong et al., 1988; Matsuoka
et al., 1988; Casey et al., 1990) and, to our knowledge, has not been
demonstrated in sympathetic neurons. However, when Western blots were
performed, Gz could not be detected with
protein from either SCG or hippocampus using any of three commercially
available antibodies (see Materials and Methods). Thus, the
RT-PCR experiment suggesting the presence of
Gz in SCG neurons could be neither confirmed
nor refuted with Western blotting experiments.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The aim of this study was to characterize the G protein coupling
profile of mGluR2, a Gi/o-coupled receptor. This
was achieved by treating mGluR2-expressing cells with PTX to inactivate
endogenous Gi/o proteins and coexpressing various
PTX-insensitive mutants of G proteins with G to reconstitute
coupling, measured as the degree of calcium current modulation upon Glu
application. In addition, RT-PCR was used to detect messenger RNA
coding for various G proteins endogenously expressed in SCG neurons.
The RT-PCR results were confirmed with Western blots where possible.
Although mGluR2 is known to couple exclusively to the
Gi/o family of G proteins, a comprehensive
characterization of coupling with members within this family has not
been reported. Such data may shed light on a potential source of
specificity, particularly in systems in which several G protein-coupled
receptors are known to coexist. For example, the existence of a subset
of G proteins in a nerve terminal coupled with the knowledge of G
protein coupling capabilities of the expressed receptors could lead to
a more complete understanding of the specific roles of individual
receptors. To date, tools are unavailable to distinguish between the
individual members of every G protein family, but
there is some evidence that G proteins may be selectively localized
in nerve terminals. In the large calyx preparation of the chick ciliary
ganglion, several G proteins have been shown to express in the
synaptic terminals and associate with the active site, whereas other
G proteins (namely Gs and
Gz), known to express elsewhere in the cells,
are excluded from close association with the membrane at release sites
(Mirotznik et al., 2000). If similar selective localization of G
proteins within a family also occurs, then this in combination with
unique receptor/G protein coupling profiles may underlie the specific
physiological roles of the receptors.
The data presented here demonstrate that mGluR2 can couple efficiently
to Gob, Gi1,
Gi2, and Gi3 and less
efficiently to Goa. Additionally, mGluR2 does
not seem to couple to the more divergent member of the
Gi/o family, Gz (Fong et
al., 1988; Matsuoka et al., 1988). Particularly intriguing is the
finding of selectivity between Gob and
Goa. This is surprising because there are few amino acid changes between these two splice variants and only one in
the extreme C terminus (N/K at 10 from the C-terminal end of the
mouse sequence used for expression in this study; Fig. 6C). The extreme
C terminus is believed to play an important role in receptor/G protein
interaction and is generally thought to be critical for selectivity in
receptor interactions (Hamm et al., 1988; Conklin et al., 1993). In
fact, several studies have shown that receptor selectivity can be
conveyed to G chimeras by swapping only the most distal five amino
acids (Conklin et al., 1993; Gomeza et al., 1996; Blahos et al., 1998).
Therefore, the finding that mGluR2 coupling is selective for
Gob over Goa suggests
that other regions of G may also be important in coupling to
receptors, at least to class 3 G protein-coupled receptors such as
mGluRs. The small number of amino acid changes across these variants
(of which just 15 are nonconservative changes) could provide a useful
starting point for investigation into the molecular basis for mGluR/G
protein interaction.
Although the measured signal (calcium current modulation) is
G-mediated, the identity of released from the various G subunits can be ruled out as the source of observed differences in
signal strength because the same G subunits
(G1,2) were used in
each experiment in this study. Although these subunits have been shown
to produce robust voltage dependent calcium current modulation,
specificity of signaling does not seem to come from specific G
subunit combinations (Ruiz-Velasco and Ikeda, 2000). In addition, the
two G subunits that displayed inefficient coupling with mGluR2 in
this study (Gz and
Goa) have been demonstrated to couple strongly
to endogenous receptors in this system by a previous study from this
laboratory (Jeong and Ikeda, 2000), using the same
GoaC351G and Gz
constructs that were used in the present study. Finally, the
differences in coupling specificity between the heterologously
expressed mGluR2 in this study and the endogenous 2 adrenergic
receptor may lead to speculation that differences in coupling result
from differential access to molecular scaffolds. However, because
distinct coupling profiles have been reported for various endogenously
expressed receptors (Jeong and Ikeda, 2000), this explanation is
unlikely to account for all of the observed differences in G protein
coupling across receptor types. Changes in mGluR2 expression levels
might have also influenced coupling. However, this did not seem to be
the case here. Results from each group were consistent despite the
variability in expression levels that normally results from cDNA
injection as judged by GFP expression.
A tacit assumption of these studies is that the C-to-G mutation in G
does not greatly influence receptor-G protein coupling fidelity.
However, the mutated residue lies within a region of G identified as
a critical determinant of receptor/G protein coupling (Hamm et al.,
1988; Conklin et al., 1993). Thus, the mutation may influence G protein
coupling to mGluRs. Although we cannot completely rule out this
possibility, studies of PTX-resistant G subunits indicate that
although the efficacy of partial agonists is altered, general
characteristics of coupling are maintained (Bahia et al., 1998).
Moreover, in the current study, neither the EC50
nor the maximal effect of reconstituted Gob
was significantly altered from control (Fig. 3). Thus, the strategy is
clearly useful for determining G protein coupling profiles within the
context of the required mutation. However, extrapolation of these data to native proteins requires some caution and alternative approaches will be required to definitively establish a coupling profile.
Results from this study confirm the conclusions from some recent
studies. Gomeza et al. (1996) and Blahos et al. (1998) demonstrated that G chimeras containing the N terminus of
Gq and the extreme C terminus of either
Go or Gi could couple to
phospholipase C via mGluR2, but similar
Gq/Gz chimeras could not.
These data indicate that mGluR2 is capable of coupling to variants of
Go and Gi, but not to
Gz, as was demonstrated here. It should be noted,
however, that the experiments in the Gomeza et al. (1996) and Blahos et al. (1998) papers were unable to distinguish coupling to
Goa from that of Gob, or
among Gi1, Gi2, and
Gi3. Also, the assay for coupling in those
studies was dependent on PLC activation by chimeric receptors. Therefore, only differences in G coupling resulting from sequence variations in the C-terminal 5 amino acids could be detected. It should
also be noted that under the conditions described here, mGluR2 seemed
unable to couple to similar
Gq/Go chimeras as described in the above studies (not shown).
One recent study examined coupling of several endogenously expressed
receptors in cultured hippocampal neurons using a strategy similar to
that described here (Straiker et al., 2002). Although mGluR2 was not
examined, a natively expressed group III mGluR was tested and seemed
unable to couple to the PTX-insensitive G proteins tested
(Goa, Gi1-3). However, the
authors note that the initial signal (synaptic inhibition by a group
III mGluR agonist) was small, which may have contributed to a
difficulty in reconstitution.
The RT-PCR results described above demonstrate the presence of mRNA
from rat SCG for Go,
Gi1, Gi2,
Gi3, and Gz. In
addition, mRNA coding for Gq,
G11, and Gs was
detected, although the Gq signal seemed weaker
than the other G subunits tested. These data are interesting
considering the findings regarding G protein coupling specificity of
heterologously expressed mGluR2. For example, although
Go seems to be expressed strongly in SCG
neurons, it is likely that any coupling between mGluR2 and
Go is primarily via
Gob, because
mGluR2/Goa coupling seems inefficient. In
addition, although Gz may be present in SCG
neurons, it does not seem to contribute to calcium current modulation
via heterologously expressed mGluR2 in this system. Regarding the
natively expressed 2 adrenergic receptor,
previous work has demonstrated strong coupling to
Goa, Gob,
Gi2, and Gz, but weak
coupling to Gi1 and
Gi3 (Jeong and Ikeda, 2000). This is
particularly interesting in light of the finding that all members of
the Gi/o family seem to be expressed in SCGs. The
implication of these findings is that in native neuronal systems,
signal specificity may arise, at least in part, from selective coupling
to individual members within G protein families. It should be noted,
however that although care was taken to minimize the number of glial
cells in the SCG preparation from which the RNA was isolated, it is
likely that some were present and may have contributed to results.
Therefore, future studies should be performed using RNA isolated from
single SCG neurons to confirm the results presented here.
The presence of Gz mRNA from SCG neurons was
unexpected. Previously, Gz protein has been
shown to be present in brain and some other tissues at low levels (Fong
et al., 1988; Matsuoka et al., 1988; Casey et al., 1990), but not in
sympathetic neurons. Here, we show that Gz
message is present in sympathetic neurons from the rat SCG, but we were
unable to confirm (or refute) this result by demonstrating the presence
of the Gz protein with Western blotting.
Western blots were also performed to confirm the presence of other G
proteins. However, because of the lack of specificity of most anti-G
antibodies tested (as judged by recognition of various recombinant G
proteins), many of these experiments produced less than meaningful
results. Exceptions were Gi2 (which was detected in SCG and did not recognize even the closely related Gi1 or Gi3)
Gq (although this antibody did not distinguish recombinant G11), and
Gs (Fig. 6).
In summary, the G protein-coupling profile of mGluR2 was characterized
using heterologous expression in SCG neurons treated with PTX and
reconstituting coupling to calcium currents by coexpressing PTX-insensitive Gi/o proteins with G.
mGluR2 was found to couple strongly to Gob,
Gi1, Gi2, and
Gi3, and less strongly to
Goa. No coupling with
Gz was observed. Finally, several G mRNAs
were detected in rat SCG with RT-PCR, including
Go, Gi1-3, Gq, G11,
Gs. Finally, message for
G15 was absent, as expected, because of its
unique expression in hematopoietic tissue (Wilkie et al., 1991).
| |
Acknowledgments |
|---|
We thank M. King for valuable technical assistance.
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Footnotes |
|---|
Received March 26, 2002; Accepted October 8, 2002
Portions of this work were supported by National Institutes of Health grants GM56180 and NS37615 (to S.R.I.) and NS10943 (to P.J.K.) while they were at the Guthrie Research Institute, Sayre, Pennsylvania.
Address correspondence to: Paul J. Kammermeier, Department of Neurobiology and Pharmacology, Northeast Ohio Universities College of Medicine (NEOUCOM), 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272. E-mail: pjkammer{at}neoucom.edu
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Abbreviations |
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mGluR, metabotropic glutamate receptor; PTX, pertussis toxin; RT-PCR, reverse transcription-polymerase chain reaction; SCG, superior cervical ganglion.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-S modify calcium current activation in bullfrog sympathetic neurons.
Neuron
5:
75-80[CrossRef][Medline]. subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons.
J Neurosci
18:
4521-4531 subunits.
Nature (Lond)
380:
258-262[CrossRef][Medline]. subunits.
Nature (Lond)
380:
255-258[CrossRef][Medline]. subunit Gz couples neurotransmitter receptors to ion channels in sympathetic neurons.
Neuron
21:
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) and cells expressing mGluR2 reconstituted
with PTX-insensitive G
). Each data set was
fit to the equation
Bmax[Glu]/(EC50 + [Glu]),
with Bmax fixed at 1. Data were normalized
to inhibition at 1 µM within each cell. Fits revealed
EC50 values for the control and reconstituted conditions of
1.3 and 3.8 µM, respectively. B, bar graph illustrating average
(+S.E.M.) calcium current inhibition in PTX-untreated controls
(Control), PTX-treated controls (PTX; both mGluR2-expressing), and G
), Gob (
). Number of cells is indicated in
parentheses.
